Insulin resistance in skeletal muscle is a major hallmark of type 2 diabetes (T2D) and an early detectable abnormality in the development of this disease. However, the molecular basis for the muscle insulin resistance of T2D is not fully understood. Our laboratory (preliminary data) and others have found that insulin resistant (obese and T2D) subjects have increased concentrations of lipopolysaccharide (LPS or endotoxin) in plasma. LPS is a component of the outer membrane of gram negative bacteria cell walls which induces an inflammatory response by activating toll-like receptor-4 (TLR4). It has been proposed that intestinal microbiota is the source of the excessive LPS observed in insulin resistant subjects (metabolic endotoxemia). TLR4 and inflammatory signaling pathways regulated by this receptor [inhibitor ?B kinase IKK-nuclear factor-?B (?F??) and mitogen activated protein kinases (MAPKs)] have been implicated in the pathogenesis of insulin resistance. Despite the accumulating evidence that insulin resistant subjects have increased concentrations of LPS in plasma, it is unclear whether metabolic endotoxemia causes inflammation and insulin resistance (impaired insulin signaling and insulin-stimulated glucose uptake) in muscle and whether these effects are mediated by TLR4. The proposed studies will address this gap by utilizing a model of metabolic endotoxemia in WT and TLR4-mutant mice. The overall hypothesis is that metabolic endotoxemia causes insulin resistance by activating TLR4 and signaling pathways downstream this receptor. To test this hypotheses the following specific aims will be addressed. Specific Aim 1 will determine whether high fat diet-induced insulin resistance involves increased intestinal permeability, elevated plasma levels of LPS, and whether TLR4 is required for the deleterious effects of LPS in vivo. Specific Aim 2 will determine whether an experimental elevation in circulating LPS, within a physiologic range, causes muscle insulin resistance in vivo and whether this effect is mediated by TLR4. In summary, the proposed studies will utilize the hyperinsulinemic, euglycemic clamp technique in WT and TLR4 mutant mice subjected to chronic high fat diet and acute endotoxin infusion, to provide new insights into the molecular mechanisms responsible for the pathogenesis of insulin resistance. If positive, our results would indicate that strategies aimed at lowering plasma LPS concentrations and/or blocking TLR4 will help to reduce inflammation and improve insulin action in patients with T2D. As such, our results could lead to the development of novel approaches for the treatment of this disease.